Hernandez A, Thomas R, Smith F, Sandberg J, Kim S, Chung D H, Evers B M
Department of Surgery, The University of Texas Medical Branch, Galveston, TX 77555-0536, USA.
Surgery. 2001 Aug;130(2):265-72. doi: 10.1067/msy.2001.115897.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a novel member of the tumor necrosis factor family, induces apoptosis in TRAIL-sensitive tumors through the activation of the caspase pathway. Sodium butyrate (NaBT) induces differentiation and apoptosis in certain colorectal cancers; the molecular mechanisms for these effects have not been clearly defined. The purpose of our study was to determine whether NaBT sensitizes TRAIL-resistant human colon cancer cells to the effects of TRAIL.
Human colon cancer cells (KM12C, KML4A, and KM20) that are resistant to TRAIL treatment alone were treated with TRAIL (100 ng/mL), NaBT (5 mmol/L), or a combination of these agents and harvested for total RNA and protein. Western blots were performed to assess intracellular expression of Flice-like inhibitory protein (FLIP), a caspase inhibitor. Percent-specific apoptosis, relative caspase-3 activity, and Annexin-V immunofluorescence were determined at 24 and 48 hours. Cell cycle--related gene expression was assessed by RNase protection.
Treatment with NaBT for 24 and 48 hours decreased FLIP protein expression in all cell lines. Furthermore, NaBT sensitized these resistant cancer cells to the effects of TRAIL with significant increases noted in cell death, caspase-3 activity, and Annexin-V staining compared with NaBT alone.
Our findings suggest that the reduction of FLIP protein levels by NaBT renders TRAIL-resistant human colon cancer cells sensitive to TRAIL-mediated apoptosis. The combination of TRAIL with agents (such as NaBT, which target proteins that prevent cell death) may provide a more effective and less toxic regimen for the treatment of resistant colon cancers.
肿瘤坏死因子相关凋亡诱导配体（TRAIL）是肿瘤坏死因子家族的一个新成员，通过激活半胱天冬酶途径诱导对TRAIL敏感的肿瘤发生凋亡。丁酸钠（NaBT）可诱导某些结直肠癌发生分化和凋亡；这些作用的分子机制尚未明确。我们研究的目的是确定NaBT是否能使对TRAIL耐药的人结肠癌细胞对TRAIL的作用敏感。
单独对TRAIL治疗耐药的人结肠癌细胞（KM12C、KML4A和KM20）分别用TRAIL（100 ng/mL）、NaBT（5 mmol/L）或这两种药物联合处理，然后收集细胞用于提取总RNA和蛋白质。进行蛋白质免疫印迹法以评估半胱天冬酶抑制剂类弗林蛋白酶样抑制蛋白（FLIP）的细胞内表达。在24小时和48小时时测定特异性凋亡百分比、相对半胱天冬酶-3活性和膜联蛋白V免疫荧光。通过核糖核酸酶保护法评估细胞周期相关基因的表达。
用NaBT处理24小时和48小时可降低所有细胞系中FLIP蛋白的表达。此外，与单独使用NaBT相比，NaBT使这些耐药癌细胞对TRAIL的作用敏感，细胞死亡、半胱天冬酶-3活性和膜联蛋白V染色均显著增加。
我们的研究结果表明，NaBT降低FLIP蛋白水平可使对TRAIL耐药的人结肠癌细胞对TRAIL介导的凋亡敏感。TRAIL与（如NaBT这类靶向防止细胞死亡的蛋白质的）药物联合使用可能为治疗耐药结肠癌提供一种更有效且毒性更小的方案。
